(Applicant's abstract) The principal objective of this proposal is to use T4 lysozyme as a model system to better understand the factors that determine the folding, stability, structure and function of proteins. The specific research to be accomplished includes the following: (1) An attempt will be made to simplify the protein folding problem by identifying which residues, or combination of residues, are critical for the folding and stability of T4 lysozyme. We wants to understand, not only how given residues contribute to stability, but also the relative importance of local versus non-local effects in protein folding. In other words, Dr. Matthews wants to determine the degree to which the structures of local segments of the polypeptide chain are determined by the amino acid sequence of that segment, and by the structural context provided by the rest of the folded protein. (2) The combination of scanning force microscopy with crystal-lattice-directed synthesis of defined polymers will be used to directly monitor the force-induced sequential unfolding of individual lysozyme molecules. This approach, in collaboration with C. Bustamante, will permit protein folding to be investigated in a completely new direction. (3) The body of structural, thermodynamic, and kinetic information on mutant T4 lysozymes will be used, in collaboration with S. Mayo, to test and improve computer-assisted protein design. As part of this effort, Dr. Matthews will continue to develop and to test rational methods to improve the stability of proteins. (4) Cavities within T4 lysozyme will be exploited to better understand protein-ligand interaction, to optimize rational approaches to drug selection and docking (in collaboration with B. Shoichet) and to engineer novel active sites. And (5) We will attempt to better understand the role of water in defining the structure, stability and folding of proteins.